Method for the identification of tissue-specific regulatory sequences

ABSTRACT

The invention relates to a method for the identification of regulatory sequences in genomes of micro-organisms, isolated ex vivo from infected tissues. The invention further relates to promoters in salmonellae identified by said method.

The present invention relates to a method for identifying regulatory sequences in the genome of microorganisms which are isolated from infected tissue ex vivo. The invention furthermore relates to promoters which are identified in salmonellas using this method.

DEFINITIONS

“Regulatory sequences” are understood as being those sequences which influence the expression strength of a gene product (protein). Examples of such regulatory sequences are sequences, such as promoters and binding sites for regulatory proteins, which are arranged 5′-primed of the structural gene encoding the gene product or sequences, such as terminators, which are arranged 3′-primed of the structural gene.

“Strong expression” is understood as meaning expression at a strength of more than 25 000 copies of a protein which are simultaneously present per cell (bacterium). “Weak expression” is understood as meaning expression at a strength of fewer than 4500 copies of the protein which are present simultaneously per cell (bacterium). Expression of from 4500 to 25 000 copies is described as being moderate expression.

Examples of factors which exert an influence on the strength of expression are the properties of the promoter, in particular the strength of the promoter, the properties of the Shine-Dalgarno sequence and the half-life of the protein inside the cell.

A promoter is described as being weak (medium-strength, strong) if, under given physiological conditions, it is able to bring about a weak (moderate, strong) expression of a protein.

The Shine-Dalgarno sequence in prokaryotes is a sequence which is located 1 to 20 nucleotides upstream of the start codon and is essential for initiating translation. The sequence TTTAAGAAGGAGATATACAT originates from gene 10 of phage T7 (NCBI Database gi:9627425) and generally brings about maximal translation in bacteria. Yarchuk et al. (1992) and Lee et al. (1999), for example, provide a review of the effects of mutated Shine-Dalgarno sequences.

The half-life of bacterial proteins is from a few minutes up to more than 48 hours.

“Differential gene expression” is understood as meaning the specific expression of individual genes or gene groups which is dependent on the developmental state of a cell, with this expression depending, in particular, on the external conditions and/or the host milieu in which the cell is present.

Gene Expression in Salmonella, in vitro and in vivo

In the case of host-adapted salmonella strains (e.g. serovar typhimurium in the mouse and serovar typhi in humans), salmonellas which are ingested orally penetrate, principally through the M cells of the Peyer's patches, into the intestinal wall and are taken up by phagocytes, such as dendritic cells, macrophages and neutrophils. Probably in these phagocytes, the salmonellas also reach the draining mesenteric lymph nodes and, from there, reach the liver, spleen, bone marrow, brain and other tissues by way of the blood stream. In all infected tissues, the salmonellas multiply intracellularly. If the host is unable to control the infection, multiorgan failure and death ensue.

In the case of strains which are not host-adapted, all that normally occurs is a self-healing diarrhea-associated enteritis. While the intestinal mucosa is partially damaged reversibly in this connection, more deeply lying tissues are not affected.

Both the infected host and the salmonellas have at their disposal extensive regulatory networks which are able to adapt the expression of numerous genes to the changing microenvironment (Hensel, 1998). In vitro studies using cell culture models show that salmonellas are able to express several hundred proteins, most of which have not yet been identified, differentially in dependence on the host cell line (Burns-Keliher et al., 1998). These in vitro data suggest that salmonellas may possibly be able to react, in each case in an adapted manner, to the large number of different microenvironments during the course of the infection (intestinal lumen, Peyer's patches, mesenteric lymph nodes, spleen, liver and bone marrow) (Yrlid et al., 2001; Salcedo et al., 2001). However, the few data which are available for other pathogens, such as Vibrio cholerae (Lee, Butler and Camilli, 2001) and Staphylococcus aureus (Goerke et al., 2000), show that regulatory mechanisms which are in some cases completely different from those in vitro are active in vivo. It can be assumed, therefore, that the cell culture data can only make a small contribution to understanding gene regulation during the salmonella/host interaction during an infection.

Qualitative investigations, which were not tissue-specific, into gene expression in salmonella in vivo have demonstrated that up to 100 genes are induced in infected mice (Burns-Keliher, Nickerson, Morrow and Curtiss, 1998). It has not thus far been possible to demonstrate a functional connection between particular regulatory sequences and differential gene expression in various host tissues, and such a connection is not otherwise evident from the prior art, either. It is primarily the difficulties of measuring gene expression in bacteria in infected tissue which are responsible for this deficiency. Consequently, important aspects of the pathogen/host interaction in the complex course of salmonellosis are not yet understood. Both the pathogenesis of virulent salmonellas and the activity of recombinant live salmonella vaccines depend on the salmonella/host interactions during colonization. For this reason, there is a need for a method for identifying those genes, or groups of genes, which are expressed differentially in different host tissues. Thus, for example, the question which is of importance for the activity of a live vaccine is whether salmonellas express genes during the extracellular, early phase in the intestinal lumen which are different from those which they express during the later, intracellular phase in liver macrophages. In laboratory practice, it is not possible, for reasons of expense and time, to use known methods to examine all the approx. 4600 genes of Salmonella enterica serovar typhimurium (McClelland et al., 2001) for differential in-vivo expression since no known method can be used as a method for screening for quantitative, tissue-specific expression.

Salmonella-based live vaccines consist of an attenuated salmonella strain which induces a limited infection, without disease symptoms, and an expression cassette having the foreign antigen whose expression is driven by an upstream located promoter. During the course of a limited infection, following oral administration of the live vaccine, an immune response is induced against the salmonella carrier and also against the antigen which is expressed heterologously. However, it has been found, in practice, that the expression of a foreign antigen can overburden the salmonellas to such an extent that they only induce a very slight infection during which no adequate immune response develops. It is therefore advantageous to express as little foreign antigen as possible until the salmonellas have not reached immunocompetent host tissue and only to induce the expression after that. Such expression systems which are inducible in vivo are as a rule based on differentially regulated promoters. Although there is some qualitative information about promoters which are induced differentially in vivo, quantitative expression data, which would make it possible to select an in-vivo inducible promoter which was optimal for a live vaccine, have not thus far been published.

Known Methods for Analyzing Gene Expression in vitro and in vivo, and for Screening

Modern methods for the global analysis of transcriptomes, using DNA microarrays, and proteomes, using two-dimensional gel electrophoresis or ICAT, offer optimum possibilities for investigating gene regulation in vitro. In the case of salmonellas, the complete sequencing of the genomes of several salmonella serovars forms a suitable database for these methods. However, under in-vivo conditions, these methods can only be applied in the case of extremely heavily infected tissues since it is only then that the number of bacteria is large enough to be able to separate bacterial RNA and/or bacterial proteins from the large quantities of host RNA and host proteins. These methods are not suitable for investigating tissues which are infected in the normal course of a salmonellosis or experimentally (mice, inoculum of up to 10¹⁰ CFU orally or 10⁷ CFU i.v.) (Diehn and Relman, 2001), since the number of bacteria in the tissue is too small (in the case of salmonellas, for example, 10²-10⁵ in the Peyer's patches).

Thus far, the only possibility of obtaining quantitative information with regard to the expression of genes by pathogens in vivo is that of using the recently developed real-time RT-PCR for amplifying bacterial transcripts directly from infected tissue samples (Goerke, Bayer and Wolz, 2001; Rokbi et al., 2001). While the methodology is very sensitive, it requires specific primers as well as adapted temperature cycles for each gene. It is therefore not suitable for being used in screening for genes and/or promoters having defined properties.

Attenuating mutations suggest that the affected genes are at least expressed in vivo at one particular time. An elegant screening method, signature-tagged mutagenesis (Hensel, 1998), makes it possible to rapidly identify functionally important mutations in salmonellas and many other pathogens. The method is based on individual genes being irreversibly inactivated by means of transposon mutagenesis. If genes which are essential in vivo are affected, the corresponding clones do not then multiply in the infected animal. The clones are missing in ex vivo isolates (negative selection). The clones are identified unambiguously by means of an individual tag which the transposons carry. The disadvantages of this method are that the only mutations which are found are those which already have a strongly attenuating effect individually even though many relevant genes, which are expressed in vivo, are only important for the infection when interacting with other genes (Mahan et al., 2000). In addition, the negative selection can only detect (only qualitatively) the first tissue-specific activation but not a transient activity or a repression during the course of the infection. It is not possible to perform a positive selection. It is not possible to obtain quantitative information with regard to gene expression or selection in accordance with the strength of the expression.

Differentially regulated promoters can also be analyzed by means of reporter gene assays. The reporter genes β-galactosidase (Slauch, Mahan and Mekalanos, 1994) and luciferase (Contag et al., 1995; Jacobi et al., 1998; Dunstan, Simmons and Strugnell, 1999) enable a sensitive detection to be performed in vivo. The expression is detected by means of a color reaction which is catalyzed by these enzymes in tissue homogenate and is consequently semiquantitative. Reporter genes are therefore mainly used for investigating individual gene assays which are already known. There has not thus far been any description of ex-vivo screening methods which use these reporters.

The IVET method (Mahan, Heithoff, Sinsheimer and Low, 2000), which is frequently used for investigating pathogenic microorganisms, is a special form of the reporter gene assay. The IVET method uses reporter genes which complement a lethal metabolic defect or mediate resistance to an antibiotic. Only those clones in which the reporter gene is inserted downstream of promoters which are active in vivo are able to survive in vivo (complementation of the metabolic defect or resistance to treatment with antibiotics). Resistance markers and complementation markers make it possible to perform a positive selection. Using this method, a variety of promoters which are inducible in vivo, some of which have an important role in the infection, has been found in various pathogens, including salmonellas as well. The method is based on a qualitative comparison of expression in vitro and expression in vivo. In practice, therefore, the reporter gene is frequently coupled to another marker which, for example, mediates a color reaction. The method is used, in particular, to identify promoters which give very low background expression in vitro, which is a disadvantage since many important genes are also expressed in vitro. It is not possible to obtain quantitative information with regard to gene expression in vivo (Gort and Miller, 2000).

It is only the new IVET variant RIVET (Lee et al., 1999) which makes it possible to analyze promoter activation kinetically during the infection. RIVET uses resolvase as the reporter gene, with the resolvase, on expression, selectively removing a resistance cassette from the genome. The ratio of resistant and sensitive clones is determined by plating out and used as a qualitative measure of the expression. This approach makes it necessary to use reporter constructs which are specially adapted in dependence on the in-vitro activity of the promoters employed, such that it is impossible to screen for previously unknown promoters. Because the resistance cassette is irreversibly removed, it is only activation, and not a transient activity or a repression, which can be detected. It is not possible to obtain quantitative data, either.

Furthermore, the green fluorescent protein GFP can be used in reporter gene assays. GFP does not require any cofactors and GFP-mediated fluorescence can be measured quantitatively on live samples using fluorescence microscopy and flow cytometry. Cell populations which fluoresce differently can be separated by means of fluorescence-activated cell sorting (FACS). In cell culture models, GFP has been used, for example, to identify some salmonella promoters which are activated selectively in infected host cells (Valdivia and Falkow, 1997). Thus far, this screening method has been used in vitro in cell culture infection models with a high MOI (multiplicity of infection). In contrast to cell cultures, infected tissue homogenates contain very many host cell fragments having bacterium-like scattering behavior and GFP-like autofluorescence, which it has not thus far been possible to distinguish from GFP-expressing bacteria when using FACS. This background of interfering particles has made it impossible to use GFP as a quantitative in-vivo reporter for pathogens, such as salmonellas, which are principally present intracellularly (Lee and Camilli, 2000). This method can also be used to a restricted extent in vivo under the special conditions of a highly infected tissue. The frog pathogen Mycobacterium marinum was, for example, investigated qualitatively in highly infected frog granulomas. It was not possible to determine the expression of the GFP quantitatively.

A ratiometric method which can be used to unambiguously differentiate the tissue autofluorescence from the GFP emission in individual bacteria, by measuring the fluorescence at two wavelengths, has recently been published (Bumann, 2001a). This thereby makes available a method for measuring protein expression (promoter strength) quantitatively even in such bacteria which have been isolated ex vivo and which are to be examined, without previously being cultivated, immediately after they have been isolated. This method has been used to track the activity of a few known promoters on the infection route taken by salmonellas, from the intestinal lumen, via the Peyer's patches and mesenteric lymph nodes, to the liver and the spleen. The individual promoters (e.g. psicA, pssaH, pphoP-1, pbgP and ppagC) were cloned, as transcriptional fusions, before the infection, homogenates of the lymph nodes, of the liver and of the spleen were treated with detergent and analyzed by means of 2-wavelength FACS. This made it possible, for the first time, to quantitatively track the activation and repression of some known salmonella promoters in specific tissues during the infection.

DESCRIPTION OF THE METHOD

A first aspect of the present invention relates to a method for screening bacterial genomes for new regulatory sequences and/or already known regulatory sequences having a previously unknown function.

The method makes it possible to identify and quantitatively characterize, as was not previously possible, new or known regulatory sequences which possess tissue-specific activity. All the regulatory sequences (in particular promoters) which are present in the investigated bacterium are tested collectively without any prior information about the sequences in question being required. In particular, the method is suitable for identifying regulatory sequences which effect differential expression in vivo and in vitro and preferably exhibit a ratio of in vivo expression to in vitro expression of at least about 8. These regulatory sequences can be used, for example, for producing live vaccines.

Another embodiment of the invention relates to the identification of regulatory sequences which, in addition to effecting strong expression in vivo also effect strong expression in vitro. On account of their strong in-vivo expression, these regulatory sequences are suitable for producing live vaccines. In addition, the strong in-vitro expression can improve the immune response to recombinant proteins since, in this case, the immune response can proceed in two phases. In the case of these regulatory sequences, the ratio of in-vivo/in-vitro expression is preferably from at least about 2 to at most about 6.

The method according to the invention comprises the steps of:

-   -   (a) introducing partial sequences, which are in each case         different, from the genome of a bacterial target organism into a         multiplicity of vectors, with the bacterial sequences being         inserted in operative linkage to a reporter gene, in particular         upstream of a reporter gene but also downstream of a reporter         gene,     -   (b) transforming host bacteria with the vectors from (a), with a         genome library of the bacterial target organism being obtained         in a host bacterium,     -   (c) comparing the strength of the expression of the reporter         gene in individual bacterial cells in vivo and in vitro,     -   (d) identifying bacterial cells which exhibit a predetermined         first expression strength in vivo and a predetermined second         expression strength in vitro, and     -   (e) where appropriate, isolating individual bacterial cells from         step (d).

This thereby makes available, after step (d) and/or after step (e), individual clones which contain the promoters, and/or other regulatory elements, having the desired expression properties on an expression vector. Step (c) of the method preferably comprises one or more of the following partial steps:

-   -   (ci) administering aliquots of the genome library to at least         one experimental animal,     -   (cii) isolating bacterial cells from the at least one         experimental animal after a predetermined period of infection,     -   (ciii) determining the strength of the expression of the         reporter gene in vivo in the bacterial cells obtained in (cii),     -   (civ) cultivating in vitro bacterial cells which, in step         (ciii), exhibit a predetermined first strength of expression of         the reporter gene in vivo, and     -   (cv) determining the strength of the expression of the reporter         gene in vitro in the bacterial cells cultivated in (civ).

In addition, these clones can be enriched in the genome library if the following procedural steps are also carried out:

-   -   (f) jointly cultivating in vitro at least a part of the bacteria         identified in step (d).     -   (g) administering the bacteria cultivated in (f), or an aliquot         thereof, to at least one experimental animal.     -   (h) repeating steps (c) to (d) or (c) to (e) using the         experimental animals infected in step (g).

After steps (d), (e) or (h), individual clones can be subjected to further investigation by carrying out the following procedural steps:

-   -   (i) propagating the clone in vitro.     -   (j) administering the bacteria cultivated in (i), or an aliquot         thereof, to at least one experimental animal.     -   (k) repeating step (c), or steps (c) to (d) or (c) to (e), using         the experimental animals infected in step (i).

Steps (i) to (k) are used, for example, for generating a profile of the expression of a regulatory sequence in different tissues.

Steps (a) and (b)

The genome library of a target bacterium to be investigated is prepared, in accordance with steps (a) and (b), in a host bacterium which is able to survive and propagate in vitro and in vivo under suitable conditions. In this connection, fragments of the genome of, for example, about 0.1-2 kb in length are inserted into a suitable vector, preferably upstream of the coding sequence of a reporter gene. In one embodiment, all the regulatory elements which are required for initiating transcription are removed from the sequence upstream of the reporter gene beforehand. An efficient Shine-Dalgarno sequence (e.g. TTTAAGAAGGAGATATACAT; SEQ ID No. 1) for initiating translation can be present at a distance of 4-14 nucleotides before the reporter gene. If the inserted fragments contain regulatory sequences, in particular promoters, the reporter gene can then be expressed when the regulatory sequences are activated provided the regulatory sequences are present in the correct orientation in relation to the coding sequence of the reporter gene (promoter-trap vector).

If, in an alternative embodiment, a constitutive promoter and a Shine-Dalgarno sequence are located before the reporter gene, other regulatory sequences which influence the activity of the promoter can be identified by inserting them upstream of the promoter.

The genome library can be prepared from the genomes of any arbitrary target bacteria. Any arbitrary bacteria can likewise be used as the host. Consequently, the target bacterium and the host bacterium can be the same or different. Gram-positive and Gram-negative bacteria are suitable for both purposes. It is possible to use bacteria of the Enterobacteriacae family, belonging to the genera Salmonella, Escherichia or Yersinia, and/or the species/serovars Salmonella enterica serovar typhimurium, Salmonella enterica serovar enteritidis, Salmonella enterica serovar typhi, Salmonella enterica serovar Dublin, or Salmonella enterica serovar cholerasuis. The Salmonella enterica serovar typhimurium strain SL 1344 is particularly suitable. In addition to this, bacteria which have a response-augmenting effect on the human immune system and which are licensed as foodstuff additives (probiotic bacteria), and which therefore come into consideration as (e.g. orally administerable) live vaccine carriers, e.g. the family Lactobacillae (Gram-positive), Lactococcae (Gram-positive) and the (Gram-negative) E. coli strain Nissle (1917) are suitable.

Where appropriate, the genome library can be constructed in an intermediate host. In this case, the plasmid DNA having the inserts is isolated from the intermediate host and used to transform the final host bacterium. E. coli is particularly well suited for being used as the intermediate host.

The genome library preferably comprises more than 90%, particularly preferably more than 99%, of the bacterial genome. With the size of the Salmonella genome being about 5 Mb (McClelland et al., 2001) and the size of the DNA insert being 0.5 kb, the Salmonella genome can be represented by about 20 000 clones (without redundancy but with both orientations). Using the method according to the invention, it is possible to generate libraries, in the intermediate host E. coli, which contain more than 2×10⁵ independent clones having insert-carrying plasmids. The plasmids can be isolated from this library and transformed into the final host, e.g. Salmonella enterica serovar typhimurium SL 1344. In total, it is possible to generate 10⁶ transformants in order to obtain coverage of the genome which is as complete as possible.

Preference is given to embodiments in which the library is prepared from the genome of the host in the host itself. Inserts having a length of from 500 to 1500 bp are well suited for identifying regulatory sequences. A length of from 500 to 700 bp is particularly suitable.

The vector for preparing the genome library essentially consists of elements for replication in the host (and, where appropriate, in intermediate hosts), one or more marker genes and/or reporter genes for the selection, and sequences for the cloning. Preferably, at least one of the reporter genes encodes a fluorescent protein, e.g. GFP (green fluorescent protein) or GFP variants (e.g. giving a superior fluorescence yield: enhanced GFP or EGFP, or providing UV excitation: GFPuv; see, for example, Sullivan and Kay, 1999, in particular p. 24) or red-fluorescent DsRed, and improved variants. The use of GFP as a fluorescent reporter protein has been described in detail (Sullivan and Kay, 1999). Expression vectors (in particular promoter-trap vectors) for GFP have been described in Valdivia and Ramakrishnan (2000).

GFP is generally very stable in living cells, with a half-life of more than 24 h. GFP variants having a clearly much shorter lifetime in vivo can be obtained by modifying the C terminus, resulting in the GFP being degraded more rapidly. The C terminal sequence AANDENYALAA (SEQ ID No. 2) is specifically recognized by proteases which degrade proteins specifically from the C terminus (Andersen et al., 1998). The degradation rate can be modulated by altering the last three amino acids in this sequence (Keiler and Sauer, 1996).

It is furthermore possible to use the GFP variant GFP_OVA (Bumann, 2001b) as the reporter protein, with this variant making it possible to obtain constructs which are stable even at a high expression (Example 1). There are thus far no known variants of GFP_OVA whose lifetime has been modulated. In order to detect weak promoters, GFP_OVA must be replaced by very stable GFP.mut3, because of the higher concentration which can be reached (Example 1).

Step (c)

Step (c) preferably comprises one or more of the partial steps (ci) to (cv). It is possible to use the customary methods for administering the genome library corresponding to step (ci) to an experimental animal: injection (max. 2×10⁷ CFU intravenously, 2×10⁹ CFU intramuscularly, 2×10⁹ CFU subcutaneously or 2×10⁷ CFU intraperitoneally), oral administration of 5×10¹⁰ CFU (pretreatment with 5 g of streptomycin/l in the drinking water) or nasal administration (5×10⁶ CFU). The customary auxiliary substances and carrier substances, which are known to the skilled person, are added where appropriate. The administration is preferably carried out intravenously (e.g. into the tail vein) since the highest colonization rates in the target organs (e.g. lymph nodes, spleen and liver) can be achieved in this way. Any mammals, in particular the mouse, the rabbit, the rat, anthropoid apes or calves, are suitable for use as experimental animals. It is possible, for example, to use BALB/c mice, immunodeficient mice, immunized mice or immunosuppressed IFN(R (−/−) mice.

The method can be used for developing live vaccines for humans provided use is made of bacteria which do not exhibit any pathogenicity, or only very slight pathogenicity, and which can be administered orally or nasally.

The period of infection in accordance with step (cii) is measured on the basis of the course of the infection and the target organs which are to be investigated. When using mice as the experimental animals and salmonellas as the host for the gene library, it is possible to use infection times of between 1 h and 120 h for tracking the course of the infection from the colonization of the Peyer's patches through to the colonization of the liver and spleen. In order to be able to conform to the animal protection provisions, the early and middle phase of the infection should be investigated. Terminally sick animals should not be investigated. Customary methods are used to homogenize and lyze the tissue samples corresponding to step (cii) under mild conditions such that the bacteria in the resulting single cell suspension very largely remain viable and able to propagate. The tissues which can be used are any infected tissues, in particular the intestinal lumen (i.e. the intestinal content is investigated), intestinal tissue, Peyer's patches, spleen, liver, lymph nodes, kidney, bone marrow, brain, blood, intraperitoneum, uterus, oviduct and connective tissue. The method is also suitable for identifying regulatory sequences which are active in a tissue-specific manner.

The identification in accordance with step (ciii) is effected using methods which are suitable for the respective reporter protein. It is important that the expression strength of the bacteria is measured immediately after they have been isolated ex vivo, without any significant protein degradation or significant protein neosynthesis, or growth and/or propagation having taken place. Furthermore, it is necessary to use methods which do not limit the viability of the bacteria and their ability to propagate. Methods which are able to determine the fluorescent proteins quantitatively in the live bacterium are therefore preferred. FACS makes it possible to identify and isolate individual bacteria which meet a predetermined criterion for fluorescence strength (as threshold or as a range with an upper and lower limit). This makes it possible to readily identify and isolate bacteria which are expressing a fluorescent protein. Because of the autofluorescence of the residues of the host tissue, the use of standard FACS, with, as is customarily the case, only one emission wavelength being measured, leads to unusable sorting results in the case of the bacterial suspensions according to the invention. A ratiometric method, with the measurement of two emission wavelengths (details of the method, see Bumann, 2001a), is therefore used, with this method enabling bacteria having a predetermined expression of the marker protein to be isolated cleanly. The strength of the fluorescence is the measure of the quantity of expressed protein and can be calibrated to copies of the protein per cell. The calibration can be effected by way of the extinction coefficient of the fluorescent protein or by way of a densitometric protein determination using reference samples of differing concentration, e.g. in an SDS gel.

The expression strength in step (ciii) is preferably provided as a threshold for implementing a positive selection (for clones which are expressing the gene). The individual bacterium then has to exceed the threshold in order to be selected. The individual bacteria which are isolated in accordance with step (ciii) are consequently clones which, under the chosen conditions, are expressing the reporter protein at a given minimum strength. This thereby isolates such clones in which the insertion sequence before the reporter gene contains a promoter which is active in the tissue under investigation.

The threshold for identifying strong promoters (positive selection) is understood as being at least 25 000 copies of the protein per bacterium. Other suitable thresholds can be selected between 25 000 and 250 000 copies/cell. If step (ciii) is passed through more than once (step (h) and/or step (k), see below), a threshold of 4500 copies is also suitable for the first passage.

Alternatively, the selection criterion can be a range, with a lower and upper limit, within which the expression must lie. It would be possible, for example, to use such a range to identify moderately strong promoters. Preference is given to using a range having a lower limit of 4500 and an upper limit of 25 000 copies.

If particular selection conditions are chosen from the range of possible limits, it is then additionally possible to achieve the situation where the selected bacterial clones are able to pass through the natural course of infection just as well as the host strain on its own. Under these conditions, therefore, the infectivity of these clones is no less than that of the host strain on its own. A reduction in colonization of up to 25% does not significantly restrict infectivity. A clone which is expressing about 4500-250 000 copies/cell of a recombinant protein in step (ciii) therefore meets these conditions since a decrease in colonization of more than 25% is not measured in this case. Preference is given to selecting clones which express between 25 000 and 250 000 copies/cell. Particular preference is given to clones which express between 50 000 and 250 000 copies/cell. The limits have to be lowered in the case of toxic recombinant proteins if the toxicity restricts the infectivity.

The clones which are obtained in step (ciii) are cultivated, in accordance with step (civ), under suitable conditions in vitro, preferably jointly or in aliquots. The skilled person is familiar with these suitable conditions. The strength of expression is determined, in accordance with step (cv), as previously described.

Step (d)

In accordance with step (d), the bacteria obtained from step (ciii) are subjected to a second selection step using the methods which are suitable in accordance with steps (civ) and (cv). A threshold for implementing a negative selection (clones which express the reporter gene below the threshold under the chosen conditions) is preferably determined. The threshold for negative selection is understood as being fewer than 2000 or 4500 copies/cell (in the 2-wavelength FACS). This threshold can be lowered still further where appropriate. Steps (c) and (d) consequently constitute a two-step selection.

Step (e)

In accordance with step (e), standard microbiological methods, which are known to the skilled person, can be used to readily isolate individual clones from the bacterial suspension derived from step (d).

Step (f)

The bacteria are cultivated in step (f) as in step (civ) using a method known to the skilled person.

Step (g)

The bacteria which are cultivated in (f) are administered, in accordance with step (g), as described for step (ci). Preference is given to using the same administration route and the same experimental animal species as in step (ci).

Step (h)

The repetition of the selection steps, in accordance with step (h), serves to enrich clones which possess the desired expression properties. The selection thresholds for the in-vivo and/or in-vitro expression strength(s) are modified where appropriate (see that step). In addition, it is possible to use the same parameters and methods as in the case of the preceding implementation(s) of the procedural steps.

Step (i)

The conditions under which the individual clones from steps (d) and (e) or (h) can be cultivated are known to the skilled person. Preference is given to using the conditions which were already employed in step (c).

Step (j)

The bacteria cultivated in (i) are administered, in accordance with step (j), as previously described. Preference is given to using the same administration route and the same experimental animal species as in step (ci) and/or step (g)

Step (k)

The repetition of the procedural steps, in accordance with step (k), is used to further investigate the expression properties of previously identified clones. In particular, it is possible to generate a profile of the strength of expression in different host tissues (e.g. Peyer's patches, spleen or liver) or host cell types (macrophages, neutrophiles or dendritic cells). Individual host cell types can be obtained by means of purification using standard methods, e.g. magnetic cell sorting, MACS or FACS (Yrlid, Svensson, Hakansson, Chambers, Ljunggren and Wick, 2001; Salcedo, Noursadeghi, Cohen and Holden, 2001).

Localization of the Regulatory Sequences

Another aspect of the invention relates to the regulatory sequences which are present in the inserts in the clones which are identified using the method. Standard methods known to the skilled person can readily be used to examine these clones for the insert which is present in the vector. The insert can readily be sequenced using standard methods. This makes it possible to further localize the regulatory sequences in the inserts which have been obtained. For this, steps (i), (j) and (k) are carried out using clones which contain the partial sequences. If it is assumed that the regulatory sequences do not, all in all, constitute more than between 50 and 200 consecutive bases, it is then possible to identify such a sequence in a few steps by bisecting the insert and subjecting the clone which is in each case functional to further investigation. If both subclones are no longer active, it is then assumed that the regulatory sequence is located in the middle and has been cut and further work is conducted with a construct of equal length which contains the cut sequence. In this way, a functional sequence of about 60 (200) bases would have been identified after at least three and at most six steps if the length of the insert is assumed to be 500 bp (1500 bp).

Another possibility for precisely locating the regulatory sequences consists in using the computer to make predictions. Any reading frames (i.e. sequences composed of a start codon and as many subsequent triplets as possible without a stop codon) which may possibly be present can be identified within the insert sequences. These predictions can be confirmed by comparison with the GENBANK sequences. The regulatory sequences in the inserts consequently as a rule end at the very latest at the proximal start codon. The sequences before the start codon (the intergenic regions) can be investigated further for the presence of putative promoters (transcription starts). The algorithms of Reese (1994), Reese and Eeckman (1995) and Reese et al. (1996) are suitable for this purpose. An implementation of the algorithms is available on the Internet under http://www.fruitfly.org/seq_tools/promoter.html. The results of the computer analysis in the case of the sequences according to the invention are presented in Tables 2 and 5. It is consequently possible to localize regulatory sequences, in the insertion, to the sequence between the putative transcription start and the proximal ORF. In order to avoid inaccuracies in predicting the transcription start, the sequence should be extended upstream by, e.g., 10, 20, 30, 40 or 50 bp.

Preference is given to adding a further sequence of 50, 100, 150 or 200 nucleotides upstream of the transcription start in order to reliably detect the parts of the regulatory sequences which are located within this sequence (e.g. the −10 and the −35 regions). Components of the regulatory sequences upstream of the −35 region can be offhand determined readily (e.g. as described above).

Live Vaccines

Another aspect of the invention relates to the use of regulatory sequences according to the invention for developing a live vaccine. Demonstrating expression in a tissue and a lack of expression in vitro simultaneously shows that the insert, or a partial sequence thereof, as the method according to the invention has identified it, contains at least one regulatory sequence from a bacterial genome and is sufficient for controlling the tissue-specific expression of recombinant proteins. As a consequence, the insert, or a partial sequence thereof, is at the same time suitable for effecting the tissue-specific expression of a recombinant antigen. For this, the insert, or a partial sequence thereof, is placed before the gene to be expressed. This construct is either integrated into the genome of a suitable vaccine strain or introduced into a vector which can be replicated in the vaccine strain, which is transformed into the vaccine strain. The construct can be inserted into the genome of the host cell, for example as a tandem, i.e. it is inserted close to the natural gene locus which is expressed by the regulatory sequence according to the invention. Examples of suitable antigens would be the Helicobacter antigen urease or AIDA fusions.

The regulatory sequences according to the invention can also be used by integrating a heterologous nucleotide sequence, which encodes a recombinant antigen or another heterologous protein, at the corresponding site in the genome of the host cell, e.g. of a bacterium, such that the regulatory sequence is able to bring about the expression of the heterologous nucleotide sequences in its natural functional context.

The gene which is expressed naturally by the regulatory sequence according to the invention can be inactivated by the insertion provided the gene is not essential (e.g. virK, ugd, sifB, pSLT046, phoN, iicA and stm2585A, see Table 4). In addition, the heterologous sequence can be inserted into an available operon without the expression of this sequence disturbing the expression of the sequences which are naturally present. Another possibility is to express the heterologous sequence as a fusion with the natural gene. The advantage of these different configurations is that inheritance is more stable in the chromosome than on expression vectors. The stability of the genetic configuration is an important criterion for the licensing of a recombinant live vaccine.

Because of their strong expression (in vivo, a maximum of 841 000 copies/cell when using expression vectors), the regulatory sequences according to the invention are particularly well suited for being integrated into the genome. Since generally only one, or a few, copy(ies) of the heterologous sequence is/are present in the chromosome, it has to be expected that the expression will be lower than when using expression vectors, which as a rule exhibit a relatively high copy number. Since 75 000 copies/cell are sufficient for achieving a saturating immune reaction (see Example 3), a reduction in expression of up to 90% can be accepted when using the regulatory sequences according to the invention in chromosomally integrated form. The regulatory sequences 4.5G, A.8H, 2.1F and 1.3G are therefore particularly preferred. A.8H and 2.1F contain regulatory sequences which express nonessential genes. These genes can therefore be replaced with heterologous sequences without impairing the colonizing ability of the salmonellas. While 4.5G and 1.3G have not thus far been characterized with regard to their virulence function, both are phage-associated. Since phages as a rule only have slight effects on salmonella virulence, it can be assumed that the genes which are regulated by 4.5G and 1.3G can also be replaced with heterologous sequences without this being to the detriment of the colonizing ability.

Those regulatory sequences according to the invention, for expressing recombinant proteins, which are naturally present in the human-pathogenic strains Salmonella typhimurium, S. typhi, S. paratyphi A or S. paratyphi B are of particular interest for developing a live vaccine. It is therefore advantageous to use Table 4 to select, from the group of regulatory sequences according to the invention, those sequences which are at least present in Salmonella typhimurium, S. typhi, S. paratyphi A or S. paratyphi B or in two or more thereof. The advantage is that, when an attenuated strain of Salmonella typhimurium, S. typhi, S. paratyphi A or S. paratyphi B is used as the vaccine strain, the recombinant protein is expressed under the control of the autologous regulatory sequence within the natural regulatory network. Consequently, the expression of the recombinant protein follows the activity of the regulatory sequence during the course of the infection and is easier to control and predict.

Another aspect of the invention relates to the graduated modulation of the expression. The expression strength, which, in the case of a live vaccine, should be chosen to have maximum effect, depends on antigen-specific factors, in particular toxicity, degradation rate and immunogenicity. An efficient method of selectively optimizing the activity of a recombinant live vaccine is to use an identified regulatory sequence which can bring about strong expression in vivo and to selectively attenuate the expression by means of selectively modifying the Shine-Dalgarno sequence. This makes it possible to optimize the activity of a recombinant live vaccine in a selective manner. It is sufficient to use, in addition to the wild-type pGO WT, one of five mutations, which are described here, of the phage T7 gene 10 Shine-Dalgarno sequence in the 16S RNA-complementary tetranucleotide GGAG and/or the adjacent nucleotides (see plasmids pGO WT, pGO_mut1, pGO_mut2, pGO_mut3, pGO_mut4 and pGO_mut5) in order to lower the strength of the expression predictably, and in a graduated manner, down to about 2% of the initial value. In this way, it is possible to cover the entire range which is reasonable for protein expression and/or antigen expression. Mutating the Shine-Dalgarno sequence makes it possible to selectively attenuate the strength at which any promoters are expressed.

DESCRIPTION OF NEW PROMOTERS WHICH HAVE BEEN IDENTIFIED USING THE METHOD ACCORDING TO THE INVENTION

The invention also relates to salmonella promoters which are active in vivo and which are contained in the insertions 4.5G, 1c, A.8H, 1f, 3g, 2a, 4a, 10g, 12b, A.2A, A.7A, A.9D, A.10F, A.11B, A.11H, A.12A, A.12G, CLII.3A, CLII.4C, CLII.9B, CLII.11C, CLII.12C, 3.2E, 3.4F, 3.6B, 3.9A, 3.9E, A.11A, A8.B, CLI.5A, 4.4G and A1.A (see FIGS. 3-29 and 33-37) as well as to promoters which are derived therefrom by means of mutations, e.g. by means of insertions, deletions and/or substitutions of single bases or short sequence segments, for example of up to 3 bases in length, and to their use for producing live vaccines, in particular for the expression of recombinant, preferably heterologous antigens, in live vaccines.

All the promoters which are described below are described here for the first time as regards their in-vivo expression strength. While data with regard to induction in macrophages in cell culture were already available in regard to plasmids 2a, 4a, 10g and 12b, these data did not enable any reliable prediction to be made in regard to in-vivo conditions (Beuzon, Unsworth and Holden, 2001). In the case of the particularly attractive promoters 1f and 3g as well as and in the case of promoter 1c, there have not previously been any data available at all in regard to expression in host cells, either in the case of cell culture infection models or in the case of in-vivo models. All the sequences apart from 3g are homologous with the known genome sequence of S. enterica serovar typhimurium LT2 (McClelland et al., 2001).

All of the promoters mentioned in Example 4 are likewise described here for the first time as regards their in-vivo expression strength. Those promoters which show an in-vivo/in-vitro expression ratio of at least about 8 are particularly suitable for differentially expressing recombinant proteins. These are described below in more detail. Data with regard to expression in cell culture are available for the promoters in the sequences A.11B, 2.2A, CLII.4C, 2.4A, A.9D, A.7A, 3.4F, 3.2E, 3.6B and 3.9E. Qualitative data in regard to in-vivo expression are additionally available in the case of the promoter in sequence 3.9E (Valdivia and Falkow, 1997). All of the sequences apart from 1.3G are homologous with the known genome sequence of S. enterica serovar typhimurium LT2 (McClelland et al., 2001). The promoters in sequences A.11B, CLII.9B, CLII.12C, A.2A, 2.1F and 1.3G approximately fulfill the selection criteria which are specified in Example 4 as being preferred. While the in-vitro threshold was in some cases markedly exceeded in clones A.10F, CLII.3A, 2.2A, 4.5G and A.8H, the ratio of the expression strengths was greater than 8. Consequently, in addition to the 13 sequences which fully satisfy the preferred selection criteria, these 11 regulatory sequences are also suitable for differentially expressing recombinant proteins (cf. Table 4).

In addition to the in-vivo/in-vitro ratio of the expression, the absolute strength of the in-vivo expression is also an important criterion for suitability for producing a live vaccine. The regulatory sequences which are present in the sequences A.11A, A.8B, CLI.5A, 4.4G and A.1A are particularly advantageous for producing a live vaccine since, in some cases, they show an in-vivo expression (from 95 000 to 222 000 copies/cell, see Table 4) in the target tissue which is greater than that of the regulatory sequences which fully or approximately satisfy the selection criteria. The expression ratio is at least 2 and at most 6. This is due to an in-vitro expression of from 36 000 to 59 000 copies/cell, which is greater than that of the above-described regulatory sequences apart from 4.5G. Aside from the T cell response, for which a delayed antigen expression, as is brought about by the regulatory sequences having a very low in-vitro expression, is optimal, the regulatory sequences A.11A, A.8B, CLI.5A, 4.4G and A1.A have the additional advantage that, because of the higher in-vitro expression, an initial quantity of antigen is provided in a live vaccine, with this initial quantity being crucial for forming antibodies. The strong in-vitro expression can consequently be used to induce a biphasic immune response. The sequences of these promoters are shown in FIGS. 33-37.

Plasmid 1c contains a transcriptional fusion of gfp_ova with aroQ, which encodes periplasmic chorismate mutase (Calhoun et al., 2001). Previously, nothing was known about the in-vitro and in-vivo expression of aroQ. Chorismate mutase is involved in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. Several powerfully attenuating mutations (aroA, aroC and aroD) are located in genes which are involved in the same metabolic pathway (Groisman and Ochman, 1997). It is possible that aroQ is likewise required for full virulence. On the other hand, the salmonella genome contains several ch6rismate mutase genes (aroQ, pheA and tyrA), for which reason the importance of aroQ is thus far unclear.

Plasmid 1f contains a genome fragment which is located immediately upstream of siffB. SifB is induced under a variety of in-vitro conditions; nothing has previously been known about its expression in host cells in vitro or in vivo (Miao and Miller, 2000). SifB is 30% identical with the known virulence factor SifA (see also below, plasmid 12b) and is presumably translocated into the host cell cytosol by means of a type III secretion system; however, the importance of SifB for infection is thus far unclear. The low expression strength of P_(sifB) in vitro, and the dynamics of its induction in vivo, are superior, for heterologously expressing foreign antigens, to those of all the other promoters which have previously been characterized quantitatively (previous best promoter P_(pagC): in-vitro activity in different salmonella strains 10 000-50 000 copies of GFP_OVA per cell, in vivo 150 000 to 230 000 copies per cell, Bumann, 2001b). In particular, the promoter contained in plasmid 1f has comparably low activity (fewer than 2500 copies per cell) in the logarithmic growth phase in vitro in a variety of strains of the typhimurium serovar, and also in the typhi strain Ty21a serovar, which is licensed as a live vaccine, and can be induced in late-stationary cultures, which means that constructs which contain this promoter can be used widely and can be readily tested in vitro.

The plasmid 2a contains a transcriptional fusion of gfp_ova with phoN, which encodes a PhoP-regulated acid phosphatase. While PhoN is induced in macrophages in cell culture, data on its in-vivo expression have thus far been lacking. PhoN is not required for salmonella virulence in the mouse model (Miller, Kukral and Mekalanos, 1989).

Plasmid 3g contains a transcriptional fusion of gfp_ova with a reading frame which is not present in Salmonella enterica serovar typhimurium LT2 but which possesses low homology with reading frame stm2137 (in this regard, compare McCleland et al., 2001). A high degree of homology exists with a sequence segment, which has not been further characterized, from Salmonella enterica serovar Dublin. The sequence of the genome is to be found in the National Center for Biotechnology Information (Taxonomy ID 98360, ref=NC_(—)002961, Contig UIUC_(—)98360). Nothing has previously been known about its expression in vitro and in vivo. The low expression strength in vitro, and the dynamics of the induction in vivo, are superior, for heterologously expressing foreign antigens, to those of all the other promoters which have previously been characterized quantitatively and are comparable to those of the promoter P_(sifB) (see above, plasmid 1f). This promoter also has a background expression which is generally low and high induction dynamics in vivo. Its ability to be induced in the stationary phase makes it easy to test new vaccine constructs in vitro. The importance of insert 3g for salmonella virulence is thus far unknown. The LT2 isolate, which lacks corresponding sequences has only low virulence in the mouse model (Wilmes-Riesenberg, Foster and Curtiss, III, 1997). In addition to a mutated rpoS gene, sequences, such as the 3g insert, which are present in the wild type are possibly also responsible for this. A reading frame (bp 572-375) having homology with phage invertases is located upstream of the transcriptional fusion. Insert 3g may therefore possibly be a mobile element which could be unstable in the salmonella genome. Further support for this is provided by the fact that a part of the tRNA 2 for serine (serU) (bp 22-83), with a high degree of congruence with the genome sequence, is located further upstream. tRNA genes are frequently insertion sites for mobile elements such as pathogenicity islands, for example. The high pathogenicity island (Schubert et al., 1999) which is rapidly lost during laboratory passages is, for example, inserted in the vicinity of serU in pathogenic E. coli bacteria.

Plasmid 4a contains a transcriptional fusion of gfp_ova with pagD, which encodes a cell envelope protein (Gunn et al., 1995). While PagD is induced in macrophages in cell culture, nothing has been previously known about its expression in vivo. PagD is not itself required for salmonella virulence in the mouse model since a deletion has no effect on the LD₅₀. On the other hand, transposon mutagenesis of this gene results in intense attenuation, which can presumably be explained by the fact that the interfering transposon reduces the expression of a gene (stm1243), which is possibly essential and which is located downstream of the P_(pagD) promoter.

Plasmid 10g contains a transcriptional fusion of gfp_ova with pipB from salmonella pathogenicity island V (Pfeifer et al., 1999). In an in-vitro cell culture infection model, salmonellas upregulate the expression of pipB in macrophage-like cells. There have not previously been any in-vivo data on its expression. PipB is required for Salmonella enterica serovar typhimurium to be fully virulent in the mouse model when small doses are inoculated orally.

Plasmid 12b contains a transcriptional fusion of gfp_ova with sifA. While SifA is induced in macrophages in cell culture, there have not previously been any data on its in vivo expression (Beuzon, et al., 2000). Since, however, the phenotype of SifA mutants (see below) can be detected in the spleen, it can be assumed that sifA is expressed in there (Salcedo, Noursadeghi, Cohen and Holden, 2001). SifA is secreted by the type III secretion system of Salmonella pathogenicity island II and is required for preserving the phagosomal membrane (Beuzon, et al., 2000). SifA mutants are greatly attenuated in the mouse model.

The properties of the promoters which are contained in the sequences 10.9B, 4.1A, 10.1B, 10.1A, 10.6A, A.7D, 4.4A, 4.7C, 4.8H, 10.7A, 4.1B, CLII.5C, A.2G, A.8D, CLII.2B, 10.12A, A.3H, A.1A, A.8B, 4.4G, A.11C, CLII.8C, A.4H, CLII.1B, A.7H, CLII.7B, A.3D, A.11A, CLI.5A, A.8C, CLII.5A, CLII.4A, CLII.9C, A.9E, A.11B, A.10F, CLII.3A, CLII.9B, CLII.12C, 2.2A, CLII.4C, CLII.11C, A.11H, 4.5G, A.12A, A.2A, 2.4A, A.12G, A.8H, A.9D, A.7A, 3.4F, 3.2E, 3.9A, 2.1F, 1.3G, 3.9E and 3.6B, in particular their function, the genes which they express, and their occurrence in different salmonella species or serovars, and other bacterial species, are described in Tables 4 and 5.

Three regulatory sequences from Example 2 (1c, 10g and 12b) were found once again in Example 4 (in this case, A.2A, 3.4F and 3.6B, see FIGS. 30-32). Homologous regions of the sequences exhibit slight differences from each other. Stronger expression was found in Example 4. This affects the upper threshold for the in-vivo selection rather than the lower threshold for the in-vitro selection and therefore leads to an increase in the in-vivo/in-vitro expression ratio (cf. Tab. 4 and Tab. 2). The reason for this is that residues (or relatively long residues) of the autologous gene impeded the expression of a GFP fusion protein in the clones from Example 2. It is therefore advantageous, when using the regulatory sequences according to the invention to express a recombinant protein differentially, to connect the sequence encoding this protein directly to the proximal start codon (see Table 2 and Table 5). Regulatory sequences which are present in clones listed in Table 4 and whose expression ratio is less than 8 according to the experimental results given in Table 4 could then also be suitable for eliciting differential expression.

The regulatory sequence in the insert 3.6B gives an in-vivo/in-vitro expression ratio of more than 400. This regulatory sequence is therefore particularly suitable for differentially expressing a recombinant protein. The insertion 12b regulatory sequence, which is homologous thereto and which shows an expression ratio of about 19 is also particularly suitable (cf. Table 2). This ratio can be improved by truncating at the proximal start codon.

The regulatory sequence in insert A.11A contains the promoter for UDP-glucose/GDP-mannose dehydrogenase, which is not essential for virulence. The functions of the genes which are expressed by the regulatory sequences of inserts A.8B, 4.4G and A.1A are unknown. The corresponding genes encode putative cytoplasmic proteins. The regulatory sequence of insert CLI.5A controls the expression of the Pho-P-dependent regulator mig-14, which is essential for virulence.

The invention will be further elucidated by means of the following figures and examples.

EXAMPLES Example 1 Comparison of Different GFP Variants as Reporters of Salmonella Gene Expression in Infected Tissues; Test of their Suitability for being used in the Screening Method According to the Invention

Green fluorescent protein (GFP) is a frequently employed reporter of gene expression in a large number of organisms. A long GFP lifetime leads to high concentrations, which can be measured readily but which may possibly also be a great burden to the expressing cells, at steady-state equilibrium. While GFP variants which have a short lifetime are less of a burden on the expressing cells, because of the smaller quantities of GFP at steady-state equilibrium, the correspondingly weaker fluorescence signal simultaneously impairs measurability. An important prerequisite is therefore the choice of a GFP variant which, in interaction with the expression strength of attractive promoter candidates, brings about optimal expression of GFP.

In order to select optimal GFP variants for investigations into salmonella gene expression during an infection, long-lifetime GFP (GFP.mut3, Cormack, Valdivia and Falkow, 1996) and different variants (GFP_OVA, GFP_ASV and GFP_LVA, Andersen, Sternberg, Poulsen, Bjorn, Givskov and Molin, 1998) were compared as in-vivo reporters for a strong promoter (P_(pagC)) or a weak promoter (P_(spvA)).

Plasmids pJBA27 (GFP.mut3), pJBA113 (GFP_ASV) and pJBA111 (pGFP_LVA) were digested with XbaI and HindIII. The 1 kb fragments were substituted for corresponding fragments in pMW57 (P_(pagC)-GFP_OVA) or pMW74 (P_(spvA)-GFP_OVA).

The resulting constructs were transformed into Salmonella enterica serovar Typhimurium SL1344 (a streptomycin-resistant calf-derived wild-type isolate which is virulent in the mouse model, see Hoiseth and Stocker, 1981). Female BALB/c mice, which were 8-12 weeks of age, were infected orally with approx. 3×10⁸ CFU. After 4 days, the colonization of the Peyer's patches was determined by plating out. The GFP fluorescence (copies per cell) was measured by means of 2-wavelength FACS.

The results are summarized in Table 1. The controls show that, under the given experimental conditions, a colonization rate of from 100 000 to 120 000 CFU can be anticipated. The data furthermore show that the expression of <4000 copies of GFP per cell does not impede colonization.

An expression of GFP of between 50 000 and 250 000 copies is accompanied by a reduction in colonization down to 75-85% of the control. Expression of 400 000 copies of GFP reduces colonization down to about 25% (P_(pagC)-GFP_ASV). 2 000 000 copies of GFP per cell massively impede colonization of the host tissue (<1%).

Using the strong promoter P_(pagC), a GFP expression of 237 000 and, respectively, 179 000 copies per cell, associated with a number of 90 000 and, respectively, 80 000 CFU isolated ex vivo, were measured when employing the GFP variants GFP_OVA and GFP_LVA. Since the colonization achieves at least 75% of that of the control strain, these GFP variants are consequently suitable for identifying strong and medium-strong promoters. The variant GFP.mut3 is not suitable since the number of CFU was too greatly reduced by the high expression. In order to search for medium-strong and strong promoters, it is consequently necessary to choose a GFP variant which has a shorter half-life than GFP.mut3 so as to ensure that 250 000 copies per cell is not exceeded.

The variants GFP_OVA and GFP_ASV are not suitable for identifying weak promoters since, when the weak promoter P_(spvA) was used, the numbers of copies per cell were too low (<4000) for detection. The variant GFP.mut3 achieved adequate expression under these conditions. It is consequently necessary, in order to search for weak and medium-strong promoters, such as P_(psvA), to choose a GFP variant which has a longer half-life than does GFP_OVA (e.g. GFP.mut3, which carries the wild-type C terminus, see Cormack et al., 1996), such that at least 10 000 copies/cell can be achieved. This value is higher than the detection threshold by a factor of 2.

Other GFP variants and other fluorescent proteins can be tested for their suitability for being used in the method according to the invention by reworking the example. To do this, it is only necessary to replace the sequence encoding the GFP or GFP_OVA in the given expression vectors with the sequence encoding the desired protein. In order to implement the method, it is necessary to achieve a copy number of between 10 000 and 250 000. Lower expression levels would also be adequate if more strongly fluorescing GFP variants, which might possibly become available in the future, were to be used. The promoters which are employed here, P_(pagC), as an example of a strong promoter, and P_(spvA), as an example of a weak promoter, can be used as promoters which cover this range.

Any other salmonella strains, or other bacterial species, can be used if they achieve at least a colonization rate of 75% of that of a plasmid-free control strain which is otherwise identical.

Example 2 Construction of a Salmonella Genome Library and Sorting in Accordance with GFP Expression in vivo; Selection Methods for Identifying Promoters which are Regulated in a Tissue-Specific Manner

A genome library was produced in Salmonella enterica serovar typhimurium for the purpose of obtaining information with regard to salmonella promoters which are active during the course of an infection. Mice were infected with aliquots of this library. The clones were isolated from infected mice and sorted by means of FACS.

The plasmid pGFP_OVA (Bumann, 2001a) was digested with BamHI. This digestion removed a 269 bp fragment, containing the P_(tac) promoter, from pGFP_OVA. The remaining 5.1 kb fragment was purified by gel electrophoresis and recircularized, giving rise to plasmid pMW82, having a promoterless GFP variant for detecting strong and medium-strong promoters. Plasmid pMW82 was digested with BamHI and dephosphorylated.

Genomic DNA was isolated from a 5 ml liquid culture of Salmonella enterica serovar Typhimurium SL1344. The genomic DNA was partially digested with Sau3a such that the fragments which were formed were in the main in a size range of from 0.5 to 1.5 kb. In addition, genomic fragments in a size range of 500-700 bp were also produced by shearing genomic DNA by ultrasonic. These fragments were treated with DNA polymerase in order to obtain smooth ends.

The genomic fragments and the promoter-free plasmid fragment were ligated under optimal quantity ratios (high number of transformants with few transformants at the same time having double inserts). The ligation preparations were transformed into highly competent E. coli (strain XL10, Stratagene) without a restriction system (in order to prevent restriction barriers in regard to the salmonella-specific DNA methylation pattern), with this making it possible to achieve a high diversity in the library (>10⁶). In all, libraries were produced which comprised more than 2×10⁵ independent clones having insert-containing plasmids and which consequently covered more than 99% of the salmonella genome. The plasmids were isolated from this library and transformed into Salmonella enterica serovar Typhimurium SL1344. In total, 10⁶ transformants were produced in order to keep the diversity of the plasmid library as extensive as possible.

An aliquot of the salmonella library having 10⁸ CFU was washed in endotoxin-free PBS and resuspended in PBS to a cell density of 2×10⁸ CFU/ml. 100 μl of this suspension (i.e. 2×10⁷ CFU) were injected into the tail veins of 8-12-week-old female BALB/c mice. 16 h after the infection, the mice were anesthetized and killed. The spleen was removed under sterile conditions and comminuted mechanically. The resulting suspension was lyzed with 0.1% Triton x-100 in PBS in order to release intracellular salmonellas.

Salmonellas having more than 4500 GFP_OVA copies per cell were purified by FACS (FacsSort, B&D or Vantage, B&D) using their typical green and orange emission (2-wavelength method). The detection threshold for this FACS equipment is 500 copies per cell. The detection threshold with the 2-wavelength method when using tissue samples is about 4500 copies/cell, determined by detecting against background. About 0.1-0.3% of all the clones were selected, with this corresponding to expectation (Valdivia and Ramakrishnan, 2000). The fluorescence signal was calibrated with reference samples of differing concentrations by means of a densitometric protein determination in an SDS gel (Coomassie staining).

The 60 000 clones which were obtained were cultivated as a collective in vitro. In a further step, salmonellas which expressed fewer than 2000 copies of GFP_OVA during exponential in-vitro growth in LB were isolated. The 75 000 clones which were obtained were highly redundant. They were recultivated as a collective and used for a fresh intravenous infection of mice (identical procedure to that in the first selection round). Salmonellas which contained more than 25 000 copies of GFP_OVA per cell after 24 h in the spleen were sorted and recultivated.

Single clones were examined individually for the desired expression properties (in this case: induction in the spleen). Of the 27 clones which have thus far been tested, 17 had a correct GFP_OVA expression. The genomic plasmid insert from correct clones was amplified by means of PCR using the primers “up” 5′-GGCCACGATGCGTC (SEQ ID No. 42) and “down” 5′-TACTCATATGTATATCTCCTTCTTA (SEQ ID No. 43) and typed by being digested with AluI and HpaI.

7 nonredundant inserts (designated 1c, 1f, 2a, 3g, 4a, 10g and 12b, see FIG. 1 and Table 2) were found in these 17 clones. They were partially sequenced using the “down” primer (sequence, see above). The screening method had thus found 7 regulatory sequences which act as strong promoters in the spleen but which show no activity, or only weak activity, in vitro.

Any possible reading frames (i.e. sequences composed of a start codon together with as many subsequent triplets as possible without a stop codon) which might possibly be present within the 7 sequences obtained were identified. This prediction was confirmed by comparison with the sequences from the publicly available NCBI GENBANK database, which is installed on a local blast server. In particular, use was made of the complete Salmonella enterica serovar typhimurium LT2 genome sequence (McClelland et al., 2001).

The example could be reworked with the following modifications: instead of AluI and HpaI, it is also possible to use other restriction enzymes which can be employed in the PCR buffer and whose cleavage sites usually occur frequently in the genome, e.g. MspI. If rebuffering is carried out, any restriction enzymes can be used.

Example 3 Optimizing Antigen Expression in Salmonellas by Mutating the Shine-Dalgarno Sequence

In order to determine the optimal strength of the expression of a foreign antigen in recombinant salmonella vaccines, which is on the one hand high enough for a potent immune reaction and on the other hand low enough to be of scarcely any impediment to colonization by the salmonellas, the antigen expression achieved by a strong, in-vivo induced promoter was attenuated by using mutated, suboptimal Shine-Dalgarno sequences. These mutants were synthesized by PCR using appropriately selected oligoprimers. The different Shine-Dalgarno sequences were cloned behind the strong in-vivo inducible P_(pagC) promoter and directly upstream of the model antigen GFP_OVA, which carries an ovalbumin T-cell epitope. We tracked, in vivo, the activation of, and blast formation by, the ovalbumin-specific T cells as a measure of the immune reaction in dependence on the expression strength.

Mutated versions of the efficient phage T7 gene 10 Shine-Dalgarno sequence (sequence 5′-TTTAAGAAGGAGATATACAT; SEQ ID No. 44), which, as one of the most efficient sequences known, is most frequently used, were generated by PCR using the primers “mut1, mut2, mut3, mut4 and mut5” (see Table 3) and “mut down” AGTGACAAGTGTTGGCC (SEQ ID No. 45) and pGO WT (corresponds to pP_(pagC)GO, Bumann, 2001b) as the template. Respectively, one base in the 16S RNA-complementary tetranucleotide GGAG, including the adjacent bases, was altered in the mutants. The resulting 214 bp-long fragments were digested with XbaI (cleavage site T|CTAGA) and NcoI (cleavage site C|CATGG) (yields a 191 bp-long fragment) and exchanged for the corresponding fragment in pGO WT. The corresponding plasmids pGO_mut1, pGO_mut2, pGO_mut3, pGO_mut4 and pGO_mut5 were transformed into attenuated Salmonella enterica serovar typhimurium aroA SL3261. Female, 8-12-week-old BALB/c mice were transgenically given 4×10⁶ T cells from Do11.10 mice (Murphy et al., 1990), which are transgenic for a T cell receptor which recognizes a dominant ovalbumin epitope. One day later (=day 0), the mice were immunized orally with 5×10⁸ CFU of the different salmonella strains. The in-vivo expression of GFP_OVA of the individual strains was determined on day 5 by means of 2-wavelength-FACS and using Peyer's patch homogenates. The immunogenicity of the individual strains was determined on day 7 on the basis of the ovalbumin-specific T cell blasts.

Using the six selected sequences as Shine-Dalgarno sequences makes it possible to lower the level of expression from 225 000 copies per cell (under the given conditions, in particular when using the given promoter) down to 3500 copies per cell (approx. 2%) (Table 3). It is consequently possible to cover the entire range which is meaningful for antigen expression. Modifying the Shine-Dalgarno sequences in order to increase expression does not result in any further advantage since the immune response is already saturating in this case.

Selecting from these six sequences is sufficient for reducing the level of expression in a graduated manner over a range of almost 2 powers of ten. Table 3 can be extended by reworking the example with other sequences. In this way, it would be possible to refine the gradation.

The data show that, in the case of GFP_OVA, an expression strength of approx. 75 000 copies per cell (see pGO_mut4) is sufficient to achieve a saturating immune reaction (approx. 40% of the transgenic T cells form blasts on day 7). Since 225 000 copies per cell scarcely cause any impairment of salmonella colonization when compared with a strain without plasmid (see Example 1), the concentration which is required for a saturating immune response is in a range which is very well tolerated.

If the properties of the promoter in a given target tissue are known, the GFP_OVA can then be replaced with any other antigen. A selection from the set of the original Shine-Dalgarno sequence and the five mutated sequences (e.g. original sequence, mut1, mut4 and mut5) can be used to rapidly determine, in the case of any arbitrary antigens, the relative level of expression which just elicits a maximum immune reaction by determining the colonization and immune reaction in parallel for corresponding constructs, as described in this example. For this purpose, it is no longer necessary to obtain an absolute determination of the antigen as copies/cell, which means that there is no need for the reporter gene.

Example 4 Other Differentially Regulated Promoters from Salmonella enterica Serovar Typhimurium SL 1344

An aliquot having 10⁸ CFU from a salmonella library which was prepared as described in Example 2 was washed in endotoxin-free PBS and resuspended in PBS to a cell density of _(2×10) ⁸ CFU/ml. 100 μl of this suspension (i.e. 2×10⁷ CFU) were injected into the tail veins of 8-12-week-old, female BALB/c mice. 16 h after the infection, the mice were anesthetized and killed. The spleen was removed under sterile conditions and comminuted mechanically. The resulting suspension was lyzed with 0.1% Triton x-100 in PBS in order to release intracellular salmonellas.

Salmonellas containing more than 4500 GFP_OVA copies per cell were purified by FACS (FacsSort, B&D or Vantage, B&D) using their typical green and orange emission (2-wavelength method). The detection threshold for this FACS equipment is 500 copies per cell. The detection threshold with the 2-wavelength method when using tissue samples is about 4500 copies/cell, determined by detecting against background. About 0.1-0.3% of all the clones were selected, with this corresponding to expectation (Valdivia and Ramakrishnan, 2000).

The 60 000 clones which were obtained were cultivated as a collective in vitro (plate culture in LB medium having an NaCl content which was reduced to 4 g, instead of 10 g, per 1). In a further step, salmonellas which expressed fewer than 2000 copies of GFP_OVA during exponential in-vitro growth in LB were isolated. The 75 000 clones which were obtained were highly redundant. They were recultivated as a collective and used for a fresh intravenous infection of mice (identical procedure to that in the first selection round). Salmonellas which contained more than 25 000 copies of GFP_OVA per cell after 24 h in the spleen were sorted and recultivated.

In 95 individual clones, the genomic plasmid insert was amplified by PCR using the primers “up” 5′-GGCCACGATGCGTC (SEQ ID No. 46) and “down” 5′-TACTCATATGTATATCTCCTTCTTA (SEQ ID No. 47) and typed by digesting with AluI and HpaI. The 61 nonredundant clones which were identified in this way were examined individually for the desired expression properties (in this case: induction in the spleen), with 58 having a correct GFP_OVA expression (see Table 4).

The inserts in the 58 clones were partially sequenced using the “down” primer (sequence, see above). In this way, the screening method found regulatory sequences which act as strong promoters in the spleen but which exhibit no activity, or only weak activity, in vitro.

Any possible reading frames (i.e. sequences composed of a start codon together with as many subsequent triplets as possible without a stop codon) which might possibly be present within the sequences obtained were identified. This prediction was confirmed by comparison with the sequences from the publicly available NCBI GENBANK database, which is installed on a local blast server. In particular, use was made of the complete Salmonella enterica serovar typhimurium LT2 genome sequence (McClelland et al., 2001). The sequences before the start codon (the intergenic regions) were investigated for the presence of putative promoters (transcription starts). The algorithms of Reese (1994), Reese and Eeckman (1995) and Reese et al. (1996) were used for this purpose.

The results for the 58 clones which were obtained are summarized in Table 4 and Table 5. The data are sorted in accordance with the in-vivo/in-vitro expression strength ratio. 13 of the 58 clones satisfy the preferred selection criteria precisely (CLII.4C, CLII.11C, A.11H, A.12A, 2.4A, A.12G, A.9D, A.7A, 3.4F, 3.2E, 3.9A, 3.9E, 3.6B), with the expression strength ratio being at least 12.5, as specified by the selection criteria (25 000/2000); a further six very nearly satisfy the criteria (differences in regard to the in-vitro threshold: A.11B, CLII.9B, CLII.12C, A.2A, 2.1F and 1.3G). In these cases, the expression strength ratio is at least about 8. While the in-vitro threshold was in some cases markedly exceeded in a further five clones (clones A.10F, CLII.3A, 2.2A, 4.5G and A.8H), the expression strength ratio was likewise greater than 8 because the in-vivo expression was correspondingly stronger.

Consequently, a total of 24 of the 58 clones contain salmonella-derived regulatory sequences which are able to bring about differential expression of protein in vivo and in vitro with the expression in vivo being at least eight-fold stronger than in vitro. The finding that only five of these 24 clones do not satisfy the preferred selection criteria shows that the method can be carried out successfully with great reliability. Incidentally, clones having strong in-vitro and in-vivo expression are also suitable for producing live vaccines because they are able to induce an improved two-phase immune response.

The regulatory sequences of the genes sifA (clone 3.6B), pipB (clone 3.4F) and aroQ (clone A.2A) were already identified in Example 2 (there, clones 12b, 10g and 1c) but with sequences which differ slightly. The three clones in this example give expression strengths which are markedly greater than those in Example 2. FIGS. 30-32 contain comparisons of the respective sequences. To a large extent, the positions of the intergenic regions, of the putative promoters (insofar as predicted in the two cases) and of the start codons concur. The only difference is that the intergenic region of clone 3.4F and/or of 10g, respectively, appears to contain two putative promoters (in each case, one predicted in each clone). Clones 10g and 12b differ from their homologs 3.4F and 3.6B, which express GFP more strongly, by the presence of a fragment of the autologous gene which is missing in 3.4F and 3.6B. As a result, a fusion protein, which may be folded in a different manner and could consequently be degraded more rapidly, is formed in 10g and 12b. In addition, rare codons may be formed at the fusion site, with these codons in some cases being substantially less well translated, and the residues of the autologous gene may impair translation of the GFP if the two genes are not in the same reading frame since GFP has its own start codon.

A fusion protein is also formed when expression takes place in clones 1c and A.2A, with, however, in the case of 1c containing a markedly larger proportion of the autologous protein, thereby explaining the weaker expression of 1c. For optimal expression, the regulatory sequences should therefore be truncated before the start codon.

The regulatory sequences of the genes pagD (clone 2.4A), phoN (2.2A) and sifB (clone 2.1F), and the sequence of the clone 3G (1.3G), have already been described in Example 2 (there, designated clones 4a, 2a, 1f and 3g).

The other 20 sequences are depicted in FIGS. 10-29.

The 24 regulatory sequences (Table 5, Nos. 35-58) are consequently also suitable for expressing heterologous proteins other than GFP, in particular heterologous antigens, selectively in the spleen. Consequently, these sequences are suitable for producing a live vaccine. The clone which is most suitable is clone 3.6B (sifA), which firstly exhibits very low in-vitro activity and secondly has the highest in-vivo activity.

In a further 5 clones (A.11A, A.8B, CLI.5A, 4.4G and A.1A, depicted in FIGS. 33-37, Table 4, Nos. 18, 19, 20, 28 and 29), the regulatory sequence brings about strong expression in vivo (from 95 000 to 222 000 copies/cell), with this being associated with what is likewise strong expression in vitro (36 000 to 59 000 copies/cell). The in vivo/in vitro expression ratio is at least 2 and at most 6. On account of their strong expression in vivo, these sequences are also suitable for expressing heterologous proteins other than GFP, in particular heterologous antigens, in large quantities in tissues, for example in the spleen. They are consequently likewise suitable for being used in live vaccines.

LEGENDS

FIG. 1:

Diagram of the 7 inserts which were obtained using the method according to the invention and which contain salmonella-derived regulatory sequences.

FIG. 2:

Nucleotide sequence of the expression vector pGFP_OVA (SEQ ID No. 3)

FIGS. 3-9:

Nucleotide sequences of the insertions 1c, 1f, 2a, 3g, 4a, 10g and 12b (SEQ ID No. 4-10).

FIGS. 10-29:

Nucleotide sequences of the insertions A.2A, A.7A, A.8H, A.9D, A.10F, A.11B, A.11H, A.12A, A.12G, CLII.3A, CLII.4C, CLII.9B, CLII.11C, CLII.12C, 3.2E, 3.4F, 3.6B, 3.9A, 3.9E and 4.5G (SEQ ID No. 11-30)

FIGS. 30-32:

The figures show the comparisons of sequence A.2A with sequence 1c, of sequence 3.4F with sequence 10g and of sequence 3.6B with sequence 12b. The underlinings mark the intergenic regions while the first base pairs of putative promoters are identified in bold and start codons are marked by a box.

FIGS. 33-37:

Nucleotide sequences of the insertions A.11A, A.8B, CLI.5A, 4.4G and A1.A (SEQ ID Nos. 31-35)

TABLE 1

Overview of the expression of reporter proteins (GFP) in salmonellas isolated ex vivo (from the Peyer's patches) and the numbers of CFUs isolated.

TABLE 2

Overview of the regulatory sequences isolated from salmonella.

TABLE 3

Overview of the Shine-Dalgarno sequence mutants employed. Mutations as compared with pGO WT are identified in bold (SEQ ID Nos. 36-41).

TABLE 4

The table summarizes the data for 58 clones from Example 4, sorted in accordance with the ratio of in-vivo expression to in-vitro expression.

Column 1: Name of the clone/insert

Column 2: Symbol in accordance with the nomenclature of McClelland et al. (2001)

Column 3: Gene locus corresponding to the nomenclature of McClelland et al. (2001) or www.TIGR.org

Column 4: Function in accordance with the annotation from the nomenclature of McClelland et al. (2001)

Column 5: Species/strain in which the sequence was discovered: STM (S. typhimurium), STY (S. typhi), SPA (S. paratyphi A), SPB (S. paratyphi B), SAR (S. arizonae), SBO (S. bongori), ECO (E. coli K12), ECH (E. coli 0157:H7), KPN (Klebsiella pneumoniae)

Column 6: in-vitro expression, given as copies per cell

Column 7: in-vivo expression in the spleen, given as copies per cell

Column 8: in-vivo/in-vitro expression ratio

Column 9: Notes: 1) satisfies the selection criteria precisely; 2) very nearly satisfies the selection criteria.

TABLE 5

Overview of the properties of the regulatory sequences from Table 4 (see also Table 2) which are most suitable for differential expression.

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45. Yrlid, U., Svensson, M., Hakansson, A., Chambers, B. J., Ljunggren, H. G. and Wick, M. J. (2001) In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection Infect. Immun. 69: 5726-5735. TABLE 1 CFU (±standard % CFU Expression of GFP Construct deviation) control (±standard deviation) P_(pagC)-GFP.mut3 400 ± 300 <1 2,000,000 ± 200,000  P_(pagC)-GFP_OVA 90,000 ± 30,000 86 237,000 ± 30,000 P_(pagC)-GFP_ASV 25,000 ± 10,000 24 385,000 ± 40,000 P_(pagC)-GFP_LVA 80,000 ± 20,000 76 179,000 ± 20,000 P_(spvA)-GFP.mut3 90,000 ± 30,000 86 56,000 ± 6000  P_(spvA)-GFP_OVA 100,000 ± 30,000  95 <4000 (too low) P_(spvA)-GFP_ASV 120,000 ± 30,000  114 <4000 (too low) Control SL1344 105,000 ± 30,000  100 None

TABLE 2 Putative Expression Proximal pro- of GFP ORF^(b) moters^(b) Insert^(a) (copies/cell) (start Intergenic (transcrip- Clone (size) in vitro in vivo codon) region^(b) tion start)  1c  800 bp 1500 16,000 bp 289 bp 207-288 bp 162*  1f  564 bp 1800 230,000 bp 595^(c) whole insert bp 271  2a  640 bp 3300 48,000 bp 298 bp 1-297 bp 255  3g  1.0 kb 3300 225,000 bp 750 bp 573-749 bp 736  4a  640 bp 1300 28,000 bp 466 bp 417-465 bp 422 10g  1.6 kb 1200 38,000 bp 431 bp 214-430 bp 400 12b  1.2 kb 800 15,000 bp 468 bp 134-467 bp 384 *This promoter is not located in the intergenic region; ^(a)size estimated on the basis of PCR products apart from in the case of 1f, for which the complete sequence is available; ^(b)bp positions refer to the previously known partial sequence of the insert as given in the annex; ^(c)start codon of sifB is located 31 bp downstream of the end of the insert.

TABLE 3 Sequence in the region of the Shine-Dalgarno in-vivo expression T cell Plasmid sequence (primer) copies per cell % blasts % pGO_WT

225,000 ± 12,000 100 40 ± 5 pGO_mut1

 33,000 ± 12,000 15 22 ± 5 pGO_mut2

 3500 ± 12,000¹ 2¹ 16 ± 5 pGO_mut3

103,000 ± 12,000 46 39 ± 5 pGO_mut4

 78,000 ± 12,000 35 41 ± 5 pGO_mut5

125,000 ± 12,000 56 42 ± 5 ¹: measured values below the in-vivo detection limit; values are based on estimates made with the aid of in-vitro data.

TABLE 4 in-vitro in vivo/ Clone Symbol Locus Function Species expr. Spleen in vitro Notes 10.9B smpA STM2685 small membrane protein A all nine 288,000 51,000 0.18 genomes 4.1A mgtA STM4456 P-type ATPase, Mg2+ all nine 316,000 58,000 0.18 ATPase transporter genomes 10.1B STM2329 STM2329 putative cytoplasmic STM and STY 167,000 40,000 0.24 protein 10.1A rsd STM4165 regulator of sigma D, all nine 143,000 37,000 0.26 shows activity in genomes binding to the large RNA polymerase subunit 10.6A crp STM3466 catabolite activator all nine 110,000 29,000 0.26 protein genomes (CAP), cAMP receptor protein (CRP family) A.7D rfc STM1332 O-antigen polymerase STM, STY, 100,000 29,000 0.29 SPA and SPB 4.4A ytfL STM4407 putative hemolysin-like all nine 527,000 171,000 0.32 protein genomes 4.7C ribB STM3195 3,4-dihydroxy-2- all nine 220,000 74,000 0.34 butanone-4-phosphate genomes synthase 4.8H stm4065 STM4065 putative permease from STM, STY, 276,000 123,000 0.45 the Na+:galactoside SPA and SPB symporter family 10.7A nmpC STM1572 new outer membrane STM, SPA, 68,000 34,000 0.50 protein, bacterial SPB, SAR porin (predicted) and SBO 4.1B tpx STM1682 thiol peroxidase all nine 250,000 166,000 0.66 genomes CLII.5C araC STM0104 transcription regulator all nine 45,000 33,000 0.73 (AraC/XylS family) for genomes ara operon A.2G hilD STM2875 regulatory helix-turn- all 45,000 33,000 0.73 helix protein, araC salmonellas family A.8D ybaJ STM0474 putative cytoplasmic all nine 27,000 20,000 0.74 protein genomes CLII.2B yeiU STM2213 putative permease all nine 47,000 40,000 0.85 genomes 10.12A rpoE STM2640 sigma E (sigma 24) all nine 134,000 121,000 0.90 factor of RNA genomes polymerase, responds to periplasmic stress A.3H pheA STM2667 bifunctional: all nine 45,000 46,000 1.02 chorismate mutase P; genomes prephenate dehydratase A.1A stm1672 stm1672 putative cytoplasmic STM, STY, 47,000 95,000 2.02 protein SPA and SPB A.8B STM4157 STM4157 putative cytoplasmic STM, SPB 45,000 124,000 2.76 protein and SAR 4.4G yejG STM2220 putative cytoplasmic all nine 59,000 163,000 2.76 protein genomes A.11C stm1633 stm1633 putative periplasmic STM, STY, 16,000 44,000 2.75 binding protein SPB and SBO CLII.8C mdoB STM4541 phosphoglycerol all nine 8500 25,000 2.94 transferase I genomes A.4H NT01ST0833 NT01ST0833 STM 12,000 41,000 3.42 (only TIGR) CLII.1B phoB STM0397 response regulator in all nine 14,000 50,000 3.57 2-component regulation genomes system together with PhoR, regulates pho (phosphate) regulon A.7H phoP STM1231 response regulator in all nine 12,000 42,000 3.50 2-component regulation genomes system together with PhoQ, transcribed genes which are expressed at low Mg++ concentration CLII.7B marC STM1521 putative MarC all nine 7500 31,000 4.13 transporter, multiple genomes antibiotic-resistance protein A.3D STM1583 STM1583 putative cytoplasmic STM, STY, 7300 31,000 4.25 protein SPA, SPB and SBO A.11A ugd STM2080 UDP-glucose/GDP-mannose all nine 36,000 164,000 4.56 dehydrogenase genomes CLI.5A mig14 STM2782 putative transcription STM, STY, 37,000 222,000 6.00 activator, polymixin SPA, SPB and SAR A.8C rna STM0617 RNase I, upregulation all nine 6700 47,000 7.01 during stress probably genomes represents a mechanism for rapidly converting cellular mRNA as a response to new surroundings CLII.5A ugtL STM1601 putative membrane STM, STY, 13,000 93,000 7.15 protein: homology with SPA and SPB Schizosaccharomyces chitinase, possibly involved in peptidoglycan metabolism CLII.4A yaoF STM1275 putative hemolysin all 4200 30,000 7.14 salmonellas CLII.9C prpA = PSTK STM1853 serine/threonine all 4700 37,000 7.87 protein phosphatase salmonellas A.9E PSLT046 PSLT046 putative STM 8,200 65,000 7.93 carboanhydrase A.11B slyA STM1444 transcription all nine 2900 23,000 7.93 2) regulator for genomes hemolysin (MarR family) A.10F STM2780 STM2780 homolog of pipB, STM, STY, 3100 26,000 8.39 putative pentapeptide SPA and SPB repeat (8 copies) CLII.3A stm1630 stm1630 putative inner STM, SPB 12,000 113,000 9.42 membrane protein and SAR CLII.9B stm1637 stm1637 putative inner STM, STY 2400 24,000 10.00 2) membrane protein and SPB CLII.12C STM0859 STM0859 putative transcription STM, SPB 2100 22,000 10.48 2) regulator, LysR family and SAR 2.2A phoN STM4319 nonspecific acid STM, STY, 8000 91,000 11.38 phosphatase SPA and SPB CLII.4C ssaM STM1413 from the secretion all 1700 25,000 14.71 1) system apparatus, salmonellas essential for virulence CLII.11C sseJ STM1631 secreted effector STM, SPB 1500 26,000 17.33 1) protein of the SPI2 and SAR secretion system, necessary for full virulence A.11H STM0809 STM0809 putative inner STM and SPB 1500 28,000 18.67 1) membrane protein 4.5G NT01ST5267 NT01ST5267 STM 42,000 841,000 20.02 (only TIGR) A.12A STM2585A STM2585A pagK homolog on STM, SPB 1700 34,000 20.00 1) Gifsy-1 prophages and SAR A.2A aroQ STM1269 putative chorismate all 2500 60,000 24.00 2) mutase salmonellas 2.4A PagD STM1244 PhoP-regulated all 1700 41,000 24.12 1) salmonellas A.12G STM0972 STM0972 homolog to secreted STM, STY, 1600 47,000 29.38 1) protein sopD SPA and SPB A.8H virK STM2781 virulence gene, all 17,000 532,000 31.29 homologous sequence to salmonellas virK in Shigella A.9D ssaB STM1393 from the secretion all 1400 92,000 65.71 1) system apparatus salmonellas A.7A sseA STM1397 secretion system STM, STY, 1800 132,000 73.33 1) effector, essential SPA and SPB for virulence 3.4F pipB STM1088 encoded on the all 1400 139,000 99.29 1) pathogenicity island: salmonellas SPI3, SPI2 effector 3.2E iicA STM4504 putative cytoplasmic all 1600 187,000 116.88 1) protein, is induced salmonellas intracellularly in cell culture, not essential for virulence 3.9A yjiS STM4521 putative cytoplasmic all 1500 261,000 174.00 1) protein salmonellas 2.1F sifB STM1602 secreted effector STM, STY, 2600 473,000 181.92 2) protein of SPI2 SPA and SPB secretion system, not essential for virulence 1.3G 3G not in Salmonella 2200 413,000 187.73 2) LT2 enterica genome serovar Dublin 3.9E ssaG STM1406 from the secretion all 1700 508,000 298.82 1) system apparatus, salmonellas essential for virulence 3.6B sifA STM1224 secreted effector STM, STY, 1500 666,000 444.00 1) protein of SPI2 SPA and SPB secretion system, not essential for virulence; replication in macrophages

TABLE 5 Intergenic Putative promoter^(b) Proximal ORF Clone Insert^(a) region^(b) (transcription start) (start codon)^(b) A.2A 600 149-232 no prediction 233 A.7A 444 132-395 148 396 A.8H 461  1-249  45 250 A.9D 585  1-219 135 220 A.10F 609  1-268 121 269 A.11B 591 588-591 no prediction 783^(c) A.11H 559 no intergenic 463 653 region A.12A 525  1-291 191 or 229 292 A.12G 604 256-387 309 388 CLII.3A 235  1-235 114 236 CLII.4C 386 118-176 no prediction 177 CLII.9B 543 405-542 465 543 CLII.11C 509  1-57 no prediction  58 CLII.12C 704 413-677 no prediction 678 3.2E 658 331-607 473 608 3.4F 628 465-628 485 683^(c) 3.6B 875 567-875 817 896^(c) 3.9A 405 196-401 268 402 3.9E 691 445-539 526 540 4.5G 594 487-594 504 641^(c) A.11A 551 350-551 512 586^(c) A.8B 529  1-135  94 136 CLI.5A 449  1-202 176 203 (GTG) 4.4G 566 212-566 445 603^(c) A.1A 518  1-324 294 325 ^(a)length of the inserts in base pairs (bp), estimated on the basis of PCR products in the case of clone A.2A; ^(b)positions refer to the partial sequences shown in FIG. 10-29 and 33-37; ^(c)the start codon is located downstream of the end of the insert and was deduced from the sequences published in McClelland et al. (2001) and TIGR. 

1. A promoter which is active in vivo and which is contained in the insertions 4.5G, A.8H, If, 3g, Ic, 2a, 4a, 109, 12b, A.2A, A.7A, A.9D, A.10F, A.1IB, A.IIH, A.12A, A.12G, CLII. 3A, CLII. 4C, CLII. 9B, CLII. 11C, CLII. 12C,
 3. 2E,
 3. 4F,
 3. 6B,
 3. 9A,
 3. 9E, A.11A, A8.B, CLI.5A, 4.4G or A.IA, as well as a promoter which is derived therefrom by mutation.
 2. A promoter as claimed in claim 1 which is contained in insertion 4.5G, A.8H, If, 3g, 3.6B or I2b, as well as a promoter which is derived therefrom by mutation.
 3. A promoter as claimed in claim 1, in operative linkage with a nucleic acid sequence which is heterologous with respect thereto.
 4. The use of promoters as claimed in claim 1 for producing live vaccines.
 5. The use as claimed in claim 4 for expressing recombinant antigens.
 6. The use as claimed in claim 1 in which the nucleic acid which encodes the recombinant antigen is inserted into the genome of the host cell.
 7. The use as claimed in claim 1 in which the nucleic acid which encodes the recombinant antigen is introduced into the host cell on an expression vector.
 8. A nucleic acid which contains a sequence which is selected from the sequences which are depicted in FIGS. 3-29 and 33-37, or a partial sequence thereof.
 9. A nucleic acid as claimed in claim 8, which contains a sequence which is selected from insertions 4.5G, A.8H, If, 3g, 3.6B partial sequence thereof.
 10. A nucleic acid as claimed in claim 8, characterized in that the partial sequence has a length of at least 40, preferably at least 50, and up to 200 nucleotides.
 11. A nucleic acid as claimed in claim 8, which comprises the partial sequences of the insertions commencing 200 bp before the beginning of the putative promoter according to Table 2 and/or 5 and up to the proximal start codon according to Table 2 and/or 5, as well as a promoter which is derived therefrom by mutation.
 12. A nucleic acid as claimed in claim 11, which comprises the partial sequences of the insertions commencing 150 bp before the beginning of the putative promoter according to Table 2 and/or 5 and up to the proximal start codon according to Table 2 and/or 5, as well as a promoter which is derived therefrom by mutation.
 13. A nucleic acid as claimed in claim 12, which comprises the partial sequences of the insertions commencing 100 bp before the beginning of the putative promoter according to Table 2 and/or 5 and up to the proximal start codon according to Table 2 and/or 5, as well as a promoter which is derived therefrom by mutation.
 14. A nucleic acid as claimed in claim 13, which comprises the partial sequences of the insertions commencing 50 bp before the beginning of the putative promoter according to Table 2 and/or 5 and up to the proximal start codon according to Table 2 and/or 5, as well as a promoter which is derived therefrom by mutation.
 15. A nucleic acid as claimed in claim 14, which comprises the partial sequences of the insertions commencing 40 bp before the beginning of the putative promoter according to Table 2 and/or 5 and up to the proximal start codon according to Table 2 and/or 5, as well as a promoter which is derived therefrom by mutation.
 16. A recombinant bacterium, characterized in that it contains a promoter as claimed in claim 1 in operative linkage with a nucleic acid sequence which is heterologous with respect thereto.
 17. A bacterium as claimed in claim 16, characterized in that the heterologous nucleic acid sequence directly follows the proximal start codon of the promoter.
 18. A bacterium as claimed in claim 16, characterized in that the nucleic acid sequence encodes a recombinant antigen.
 19. A live vaccine composition, characterized in that it contains a bacterium as claimed in claim 18 as well as pharmaceutically customary carrier substances or auxiliary substances, where appropriate.
 20. A live vaccine composition as claimed in claim 19, characterized in that the bacterium is selected from Salmonella typhimurium, S. typhi, S. paratyphi A or S. paratyphi B and the promoter is autologous with respect to the bacterium.
 21. A Shine-Dalgarno sequence which can be obtained by at least one mutation of the sequence TTTAAGAAGGAGATATACAT [SEQ ID NO: 1].
 22. A sequence as claimed in claim 21, characterized in that a base is replaced with a different base.
 23. A sequence as claimed in claim 22, which is selected from the sequences shown in Table
 3. 24. A sequence as claimed in claim 21 in operative linkage with a recombinant antigen.
 25. The use of a mutated Shine-Dalgarno sequence as claimed in claim 21 for modulating gene expression.
 26. The use as claimed in claim 25 for modulating the expression of recombinant antigens in live vaccines.
 27. A recombinant bacterium, characterized in that it contains a Shine-Dalgarno sequence as claimed in claim 21 in operative linkage with a nucleic acid sequence which is heterologous with respect thereto.
 28. A bacterium as claimed in claim 27, characterized in that the heterologous nucleic acid sequence directly follows the proximal start codon of the promoter.
 29. A bacterium as claimed in claim 27, characterized in that the nucleic acid sequence encodes a recombinant antigen.
 30. A live vaccine composition, characterized in that it comprises a bacterium as claimed in claim 29 as well as pharmaceutically customary carrier substances or auxiliary substances, where appropriate.
 31. A live vaccine composition as claimed in claim 30, characterized in that the bacterium is selected from Salmonella typhimurium, S. typhi, S. paratyphi A or S. paratyphi B and the promoter is autologous with respect to the bacterium.
 32. A method for identifying regulatory nucleic acid sequences in the genome of a bacterial target organism, comprising the steps of: (a) introducing partial sequences, which are in each case different, from the genome of the target organism into a multiplicity of vectors, with the bacterial sequences being inserted in operative linkage to a reporter gene, (b) transforming host bacteria with the vectors from (a), with a genome library of the bacterial target organism being obtained in a host bacterium, (c) comparing the strength of the expression of the reporter gene in individual bacterial cells in vivo and in vitro, (d) identifying bacterial cells which exhibit a predetermined first expression strength in vivo and a predetermined second expression strength in vitro, and (e) where appropriate, isolating individual bacterial cells from step (d).
 33. The method as claimed in claim 32, characterized in that step c) comprise at lease one of the partial steps: (ci) administering aliquots of the genome library to at least one experimental animal, (cii) obtaining bacterial cells from the at least one experimental animal after a predetermined period of infection, (ciii) determining the strength of the expression of the reporter gene in vivo in the bacterial cells obtained in (cii), (civ) cultivating in vitro bacterial cells which, in step (ciii), exhibit a predetermined first strength of expression of the reporter gene in vivo, and (cv) determining the strength of the expression of the reporter gene in vitro in the bacterial cells cultivated in (civ).
 34. The method as claimed in claim 32, which furthermore comprises: (f) jointly cultivating in vitro at least a part of the bacterial cells identified in step (d), (g) administering bacterial cells cultivated in step (f) to at least one experimental animal, and (h) repeating steps (c) to (d) or (c) to (e) with the experimental animals infected in step (g).
 35. The method as claimed in claim 32, which furthermore comprises: (i) cultivating in vitro at least a part of the bacterial cells identified in step (e) or (h) (j) administering bacterial cells cultivated in step (f) to at least one experimental animal, and (k) repeating steps (c) to (d) or (c) to (e) with the experimental animals infected in step (j).
 36. The method as claimed in claim 32, characterized in that the bacterial sequences are inserted upstream of the reporter gene.
 37. The method as claimed in claim 32, characterized in that the regulatory sequences are selected from promoters and regulatory nucleic acid sequences which affect the activity of a promoter.
 38. The method as claimed in claim 37, characterized in that use is made, for identifying promoters, of a vector from which the sequences which are required for initiating transcription of the reporter gene have been removed.
 39. The method as claimed in claim 37, characterized in that use is made, for identifying regulatory sequences which affect the activity of promoters, of a vector which contains a constitutive promoter for expressing the reporter gene.
 40. The method as claimed in claim 32, characterized in that use is made of target bacteria which are suitable for being used as live vaccine carriers.
 41. The method as claimed in claim 40, characterized in that the target bacteria probiotic bacteria employed are salmonellas or bacteria.
 42. The method as claimed in claim 32, characterized in that the genome library is initially prepared in an intermediate host and then isolated therefrom and used for transforming the host bacterium.
 43. The method as claimed in claim 32, characterized in that use is made of a reporter gene which encodes a fluorescent protein.
 44. The method as claimed in claim 32, characterized in that the experimental animal employed is a nonhuman mammal.
 45. The method as claimed in claim 32, characterized in that bacterial cells which exhibit a first expression strength in vivo of at least 4500 copies per cell are selected.
 46. The method as claimed in claim 45, characterized in that bacterial cells which exhibit a first expression strength in vivo of from 4500 to 25 000 copies per cell are selected.
 47. The method as claimed in claim 46, characterized in that bacterial cells which exhibit a first expression strength in vivo of from 25 000 to 250 000 copies per cell are selected.
 48. The method as claimed in claim 32, characterized in that bacterial cells which exhibit a second expression strength in vitro of fewer than 4500 copies per cell are selected.
 49. The method as claimed in claim 48, characterized in that bacterial cells which exhibit a second expression strength in vitro of fewer than 2000 copies per cell are selected.
 50. The method as claimed in claim 32, characterized in that the identified sequences are localized further.
 51. The use of the regulatory sequences obtained by the method as claimed in claim 32 for producing live vaccines.
 52. The use as claimed in claim 51 for expressing recombinant antigens.
 53. The use as claimed in claim 52 in which the nucleic acid which encodes the recombinant antigen is inserted into the genome of the host cell.
 54. The use as claimed in claim 52 in which the nucleic acid which encodes the recombinant antigen is introduced into the host cell on an expression vector.
 55. The use as claimed in claim 50, characterized in that the expression strength of the regulatory sequences is modulated.
 56. The use as claimed in claim 55, characterized in that the modulation is effected by means of mutations in the Shine-Dalgarno sequence.
 57. The use as claimed in claim 55, characterized in that the modulation comprises a reduction in expression strength of down to 2% of the initial value. 